This invention relates to improved chemiluminescent 1,2-dioxetane compounds. More particularly, this invention relates to improved enzymatically cleavable chemiluminescent 1,2-dioxetane compounds that contain enzymatically removable labile groups. Such labile groups prevent the molecule from decomposing to produce light, i.e visible light or light detectable by appropriate instrumentation, until an appropriate enzyme is added to remove the labile group.
One enzyme molecule can affect the removal, through a catalytic cycle, of its complimentary labile group from thousands of enzymatically cleavable chemiluminescent 1,2-dioxetane molecules. This is a marked contrast to the situation with chemically cleavable chemiluminescent 1,2-dioxetanes, where one molecule of chemical cleaving agent is needed to remove the complimentary labile group from each dioxetane molecule.
Enzymatically cleavable light-producing 1,2-dioxetane compounds will usually also contain stabilizing groups, such as an adamantylidene group spiro bonded to the dioxetane ring""s 3-carbon atom, that will aid in preventing the dioxetane compound from undergoing spontaneous decomposition at room temperature (about 25xc2x0 C.) before the bond by which the enzymatically cleavable labile group is attached to the remainder of the molecule is intentionally cleaved. Wierynga, et al., Tetrahedron Letters, 169 (1972), and McCapra, et al., J. Chem. Soc., Chem. Comm., 944 (1977). These stabilizing groups thus permit such dioxetanes to be stored for exceptionally long periods of timc before use, e.g., for from about 12 months to as much as about 12 years at temperatures ranging from about 4xc2x0 C. to about as much as 30xc2x0 C. without undergoing substantial decomposition.
This invention further relates to the incorporation of its dioxetane molecules in art-recognized immunoassays, chemical assays and nucleic acid probe assays, and to their use as direct chemical/physical probes for studying the molecular structure or micro structures of various micro molecules, synthetic polymers, proteins, nucleic acids, catalytic antibodies, and the like, to permit an analyte-to chemical or biological substance whose presence, amount or structure is being determined to be identified or quantified.
Applications naming one or more of the inventors herein, as inventors, and assigned to Tropix, Inc., have clearly established 1,2-dioxetanes as chemiluminescent compounds which can be used as reporters and labels in ultra sensitive assays that can be conducted quickly, without resort to exotic conditions or elaborate apparatus, for the detection of a variety of biological materials. Among these are U.S. Pat. Nos. 4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978,614; 5,032,381; 5,145,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900; 5,336,596; and 5,871,938. All of the foregoing are incorporated herein by reference. Other patents commonly assigned with this application have issued, and other applications are pending. Together this wealth of patent literature addresses 1,2-dioxetanes, stabilized by a typically polycyclic group, such as spiroadamantane bonded to one of the carbons of the dioxetane ring, and a moiety bonded to the remainder carbon of the dioxetane ring which is electron sensitive, such that the protection of the electron sensitive moiety, typically an aryl group, leads to an anion, generally an oxyanion, which is unstable, and decomposes. Through decomposition, the 0xe2x80x940 bond is broken and a photon is generated. The same carbon atom to which this electron sensitive moiety is bonded may bear an alkoxy or other electron-active group.
The first of the dioxetanes of this class commercialized was 3-(4-methoxy-spiro(1,2-dioxetane-3,2xe2x80x2-tricyclo(3.3.1.13,7) decan)-4-yl)phenyl phosphate, particularly the disodium salt, generally known as AMPPD(copyright). This compound has been commercialized by assignee of this application, Tropix, Inc., as well as a company of Detroit, Mich., Lumigen, Inc. Superior performance of the above described compounds can be obtained by selective substitution on the spiroadamantane ring. Substitution, at either bridgehead carbon with an electron active species, such as chlorine, improves reaction speed and signal to noise ratio (s/n). The chlorine substituted counterpart of AMPPD(copyright), CSPD(copyright), has been widely commercialized by Tropix, Inc. of Bedford, Mass. xe2x80x9cThird-generationxe2x80x9d dioxetane compounds of similar structure, wherein the aryl moiety also bears an electron active substituent, such as chlorine, offer further improvements in performance, and have been commercialized by Tropix, Inc. The phosphate moieties are available under the trademarks CDP(copyright) and CDP-Star(copyright).
However, it has been observed that AMPPD(copyright) in aqueous solution, and also in the presence of chemiluminescent enhancers, e.g., a polymeric ammonium, phosphonium or sulphonium salt such as poly[vinyl benzothiazole(benzothiazole dimethyl ammonium chloride)] (xe2x80x9cBDMQxe2x80x9d) and other hetero polar polymers may exhibit longer than optimum periods of time to reach constant light emission characteristics (xe2x80x9ct xc2xdxe2x80x9d, defined as the time necessary to obtain one-half of the maximum chemiluminescence intensity at constant, steady-state light emission levels; this emission half-life varies as a function of the stability of the dioxetane oxyanion in various environments).
Statistically, approximately seven t xc2xd periods are required to reach steady-light emission kinetics. The t xc2xd of AMPPD(copyright) at concentrations above 2xc3x9710xe2x88x925 M in an aqueous solution at pH 9.5 in the presence of BDMQ have been found to be 7.5 minutes. At 4xc3x9710xe2x88x923 M in the absence of BDMQ, the t xc2xd has been found to be approximately 30-60 minutes, while at 2xc3x9710xe2x88x925 M in an aqueous solution, the t xc2xd for AMPPD(copyright) has been found to be 2.5 minutes.
In rapid bioassays that employ enzymatically cleavable chemiluminescent 1,2-dioxetanes as reporter molecules, it is desirable to reach steady-state light emission kinetics as quickly as possible so as to detect an xe2x80x9cendpointxe2x80x9d in the assay. While chemiluminescent intensity can be measured before achieving steady state kinetics, sophisticated, thermally controlled luminometry instrumentation must be used if one wishes to acquire precise data prior to steady-state emission kinetics.
Furthermore, AMPPD(copyright), in an aqueous buffered solution both in the presence and absence of chemiluminescent enhancers such as BDMQ, exhibits higher than desirable thermal and non-enzymatically activated light emission, or xe2x80x9cnoisexe2x80x9d. Such noise can be attributed to emission from the excited state adamantanone and of the methyl m-oxybenzoate anion derived from the aromatic portion of the AMPPD(copyright) molecule. This noise can limit the levels of detection, and thus prevent the realization of ultimate sensitivity, as the measured noise level of AMPPD(copyright) is approximately two orders of magnitude above the dark current in a standard luminometer.
Importantly, various instruments for detecting chemiluminescent emission such as CCD cameras have greater detection sensitivities in the green and red wavelengths. AMPPD(copyright) and related dioxetanes typically emit in the blue wavelengths of the visible spectrum. Heretofore it has been necessary to use polymeric enhancers to xe2x80x9cshiftxe2x80x9d the emission wavelength. It would be desirable to obtain dioxetanes which emit in wavelengths closer to the xe2x80x9cred or green endxe2x80x9d of the visible spectrum, to enhance detection sensitivity.
It is, therefore, an object of this invention to decrease the time necessary to conduct assays, and particularly bioassays, in which enzymatically cleavable chemiluminescent 1,2-dioxetanes are used as reporter molecules.
It is also an object of this invention to provide new and improved enzymatically cleavable chemiluminescent 1,2-dioxetanes which, when used as reporter molecules in assays, and in particular bioassays, reduce the time required to complete the assay.
A further object of this invention is to provide a new and improved enzymatically cleavable chemiluminescent 1,2-dioxetane for use as substrates for enzyme-based assays, and particularly bioassays, which provide improved signal to background behavior and thus provide improved detection levels.
A further object of this invention is the provision of dioxetane whose emission wavelengths are shifted toward the green and red wavelengths.
A still further object of this invention is to provide novel intermediates useful in synthesizing these improved enzymatically cleavable 1,2-dioxetanes.
Another object of this invention is to provide methods of preparing these enzymatically cleavable chemiluminescent 1,2-dioxetanes and intermediates thereof.
These and other objects, as well as the nature, scope and utilization of this invention, will become readily apparent to those skilled in the art from the following description and the appended claims.
The above objects, and others, made clear by the discussion set forth below, is met by a new family of dioxetanes of the general formula(s): 
wherein each R may independently be any branched alkyl or cycloalkyl group which provides stabilization for the dioxetane, or both R groups may be joined in a cycloalkyl or polycycloalkyl moiety Spiro bound to the dioxetane ring wherein the R group or groups may be unsubstituted or substituted with a halogen atom, an alkoxy group, or an electron-withdrawing organic group, and wherein R1 is an aryl group, or an alkyl group of 1-20 carbon atoms, which may be optionally substituted with 1 or more halogen atoms, and wherein Y may be H, or an electron donating or withdrawing group, or an organic linker group attached to an ancillary fluorophore, or to any biological moiety, and wherein X may be any protecting group which is removable by chemical or enzymatic means, wherein R2-R6 are independently H, alkyl, or branched alkyl,substituted alkyl, aryl, substituted aryl, wherein R3 and R4 may be joined as a spiro-fused cycloalkyl group.
The above dioxetanes may be synthesized by way of the fused benzothiazole aldehyde, or toluene derivative using permanganate oxidation to provide a carboxylic acid. Esters of this acid may be used to prepare dioxetane precursors for the above compounds according to methodology described in U.S. Pat. No. 5,731,445 which is incorporated herein by reference.